A. Genetic analyses of TP53, RAS and CDKN2 in 11 metachronous lung cancer of patients surviving at least 2 years after small cell lung cancer have found 7 mutations (4 in TP53, 2 RAS, and 1 CDKN2). Three of these mutations are G:C to T:A transversions on the coding strand. A similar analysis of 5 NSCLC tumors occurring in patients surviving Hodgkin's disease found 2 TP53 mutations (a (G:C to T:A transversion and a 1 bp deletion). The frequency and pattern of these mutations is similar to the pattern of mutations found in all lung cancers and suggesting that tobacco smoke plays a role in the development of these metachronous tumors. B. Homozygous deletions of CDKN2 were more commonly in cell lines from patients with advanced stage [13 of 46 (28%) from patients with stage Ill or IV compared to 0 of 10 from patients with stage I or II]. Intragenic mutations were detected by PCR-SSCP only in cell lines [6 of 71(8%)] and tumors [4 (7%)] from patients with advanced disease. Two patients with no mutation in their primary tumor had a CDKN2 point mutation detected in a metastatic tumor. Point mutations were G:C to T:A transversion on the coding strand in 5 of 10. No intragenic mutations of MTS2 were identified by SSCP in the 125 samples. Thus CDKN2 is a frequent target of mutation in NSCLC. These mutations are associated with tumor dissemination and have a pattern similar to that found in TP53 in smoking-related tumors. C. Among 11 cervical cell lines (including 8 HPV positive cell lines, 2 HPV negative cell lines containing mutant Rb, and one tumorigenic cell line derived from normal cervical cells following transfection with HPV-16 and v-H-ras), no homozygous deletion nor intragenic mutations were identified. Thus, CDKN2 alterations are uncommon among tumor cells with compromised Rb activity including HPV-positive cell lines.